The perinatal switch from fetal hemoglobin (Hb F,alpha2gamma2) to adult hemoglobin (alpha2beta2) is incomplete, variable, and reversible. Hb F persist in a subset of erythrocytes called F cells, whose production is controlled by factors linked and unlinked to the beta-globin gene region. The F Cell Production (FCP) Locus, is X-linked and accounts for about 40% of the variation in Hb F level in sickle cell (SS) disease. Increases in F cell production can ameliorate the clinical severity of SS Disease and thalassemia. Three classes of agents (cell-cycle specific agents, hematopoietic growth factors, and fatty acid analogues) can increase F cell production to a variable extent. This proposal is directed toward understanding the genetic mechanism(s) which account for the variation in baseline F cell levels and the variable response to switching agents. Using positional cloning techniques we will localize the FCP locus to approximately 1 million base pairs within or near Xp 22.2. We then will identify conserved sequences within this area using cosmids and Yeast- Artificial-Chromosomes which span this region. We will test these sequences for potential FCP function by screening cDNA expression libraries from hematopoietic stem cells, erythroid progenitors and human bone marrow. Candidate genes will be tested using segregation analysis between polymorphic sites in the genes and the FCP phenotype among SS male siblings and their families. Expression of candidate gene function will be screened using reverse-transcriptase polymerase chain reaction to probe mRNA from differentiating erythroid progenitors and precursors and by transfection of sense and antisense sequences into hematopoietic stem (CD34+) cells. Since transgenic mice containing the beta-globin gene region produce F cells, our most likely candidate genes will be tested in this animal model. Clues to the function of the FCP gene product will be pursued by determining if F cell progenitors have altered cell cycle kinetics in erythroid culture. These experiments may offer important clues to the kinds of proteins that might be expected to be produced by the FCP locus, thereby aiding our evaluation of candidate genes. Finally, we will continue to screen promising switching agents in vitro and in vivo to determine which agent or combination of agents maximally induce F cell production in man. This comprehensive and complementary approach to the problem of F cell production (identification of genetic controls, understanding how F cells and non-F cells differ during differentiation, and testing agents which increase F cell production) has in the past and will continue to offer the most likely chance for developing effective therapeutic agents for the beta-hemoglobinopathies.